Iron-reducing strain capable of degrading petroleum hydrocarbon under anaerobic condition and application of iron-reducing strain

Abstract

The invention belongs to the technical field of environmental biology, and particularly relates to an iron-reducing strain capable of degrading petroleum hydrocarbon and application of the iron-reducing strain. The strain is Citrobacter amalonaticus Z1 which takes petroleum hydrocarbon as an electron donor, and the Citrobacter amalonaticus Z1 is preserved in the China Center for Type Culture Collection on October 12, 2023, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 20231863. The strain Z1 provided by the invention has a degradation effect on petroleum hydrocarbon in a ferric citrate culture medium under an anaerobic condition, particularly under the coexistence condition of sodium lactate as a substrate, sodium lactate and petroleum hydrocarbon can be jointly used as an electron donor and a carbon source, the strain has a better degradation effect on petroleum pollutants, and meanwhile, ferric iron is used as an electron acceptor, so that the degradation efficiency of petroleum pollutants is improved. And reducing the iron into ferrous iron. In anaerobic remediation of petroleum-contaminated soil or underground water, the remediation microbial inoculum constructed by using the iron-reducing bacteria has popularization and application feasibility, and especially has a wide application prospect in in-situ remediation of contaminated soil in a natural ecological system.

Description

Technical Field:

[0001] The present invention belongs to the field of environmental biotechnology, and particularly relates to an iron-reducing strain capable of degrading petroleum hydrocarbons and its application. Background Art:

[0002] In China, the oilfield exploitation area is wide and the annual crude oil production is large. During the processes of crude oil exploitation, gathering, transportation, storage and refining, large areas of soil have been polluted. The physical and chemical properties and components of petroleum-polluted soil will change, and after toxic and harmful substances enter the soil, they may endanger other organisms and human health through the food chain. Among the components of petroleum hydrocarbons, alkanes are the components with the highest content in petroleum hydrocarbons. According to the structure, alkanes can be divided into straight-chain alkanes, branched-chain alkanes and cycloalkanes. In the components of most oilfield-polluted soils, straight-chain alkanes account for a relatively large proportion.

[0003] Microbial remediation is a commonly used method for soil pollution treatment at present. It has the characteristics of low cost, high efficiency and environmental friendliness, and is widely used in soil pollution remediation. Microbial remediation is generally divided into two categories. One is aerobic biodegradation, mainly under natural or artificial oxygen supplementation conditions, using aerobic bacteria to degrade petroleum hydrocarbons; the other is anaerobic biodegradation. Under anoxic conditions, anaerobic bacteria use petroleum hydrocarbons as electron donors to complete the metabolism of pollutants. Especially for soils and groundwater that have been polluted for a long time in the natural environment, pollutants generally migrate and penetrate with runoff and other effects, and are buried relatively deep. The environment where pollutants are located is mostly anoxic or anaerobic. Therefore, for the degradation of these pollutants, anaerobic bacteria generally play a major role.

[0004] During the anaerobic degradation process of microorganisms, petroleum hydrocarbons are used as electron donors. Correspondingly, ferric iron is used as an electron acceptor. Iron is a redox-active metal, accounting for 4.7% of the earth's crust content and widely existing in the soil. Iron has two oxidation states: ferrous iron and ferric iron. Iron-reducing bacteria are large in number, diverse in species and wide in distribution in the soil. Iron-reducing bacteria are microorganisms that use Fe(III) as an external electron acceptor during metabolism and reduce Fe(III) to Fe(II). Using petroleum hydrocarbons as electron donors, they can degrade petroleum hydrocarbon substances in an anaerobic environment, so as to achieve the remediation of petroleum hydrocarbon-polluted water bodies and soils. Summary of the Invention:

[0005] The purpose of the present invention is to provide an iron-reducing strain capable of degrading petroleum hydrocarbons under anaerobic conditions and its application.

[0006] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0007] An iron-reducing strain capable of degrading petroleum hydrocarbons under anaerobic conditions, the strain is Citrobacter amalonaticus Z1 which uses petroleum hydrocarbons as an electron donor, and it was deposited at the China Center for Type Culture Collection on October 12, 2023, with the deposit number CCTCC NO: M 20231863.

[0008] Application of the iron-reducing strain capable of degrading petroleum hydrocarbons, the application of the strain in the iron reduction reaction.

[0009] The application of the strain in degrading petroleum hydrocarbons in the environment.

[0010] The application of the strain in degrading petroleum hydrocarbons in soil or groundwater under anaerobic conditions, using ferric iron as an electron acceptor and small-molecule straight-chain or branched-chain alkanes as electron donors.

[0011] A bacterial agent, the bacterial agent contains the Citrobacter amalonaticus Z1 described above.

[0012] The bacterial agent contains the cells, cultures, concentrates of cultures or cell suspensions of cultures of Citrobacter amalonaticus Z1.

[0013] An application of the bacterial agent described above, the application of the bacterial agent in degrading petroleum hydrocarbon pollutants in environmental media using ferric iron as an electron acceptor under anaerobic conditions.

[0014] The application of the bacterial agent in in-situ degradation of small-molecule straight-chain or branched-chain alkanes in water bodies and / or soil.

[0015] Advantages of the present invention:

[0016] The strain Citrobacter amalonaticus Z1 provided by the present invention, which can reduce ferric iron and degrade petroleum hydrocarbons, has good tolerance to petroleum hydrocarbons and can participate in the iron cycle process in nature. Citrobacter amalonaticus Z1 is a new type of anaerobic Citrobacter, which supplements microbial resources and provides a promising bacterial agent remediation solution for the environmental problem of petroleum hydrocarbon pollution in soil. Description of the drawings:

[0017] Figure 1 A phylogenetic tree constructed by homology comparison of the 16S rRNA gene sequences of the iron-reducing strain Citrobacter amalonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention and related strains;

[0018] Figure 2Growth curve of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention under different pH conditions;

[0019] Figure 3 Growth curve of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention under different temperature conditions;

[0020] Figure 4 Growth graph of ferrous iron concentration of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention under different carbon source conditions;

[0021] Figure 5 Degradation performance graph of n-hexadecane of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention under 40-day culture;

[0022] Figure 6 Iron reduction performance graph of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention under 40-day culture;

[0023] Figure 7 Growth trend graph of total Fe(II) in soil of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention under 40-day culture;

[0024] Figure 8 Trend graph of n-hexadecane degradation in soil of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading n-petroleum hydrocarbons of the present invention under 40-day culture;

[0025] Figure 9 Graph of percentage change of different forms of Fe(II) in total Fe(II) in soil of iron-reducing strain Citrobacter malonaticus Z1 capable of degrading petroleum hydrocarbons of the present invention under 40-day culture. Detailed implementation manners:

[0026] The technical solutions of the present invention will be further specifically described below through specific embodiments, but the present invention is not limited to these embodiments.

[0027] The strain Z1 provided by the present invention can degrade petroleum hydrocarbons in a ferric citrate medium under anaerobic conditions. Especially in the coexistence of sodium lactate as a substrate, sodium lactate and petroleum hydrocarbons can jointly serve as electron donors and carbon sources, and the degradation effect of this strain on petroleum pollutants is better. At the same time, ferric iron is used as an electron acceptor and reduced to ferrous iron. In the anaerobic remediation of petroleum-contaminated soil or groundwater, the remediation agent constructed by iron-reducing bacteria has the feasibility of popularization and application, especially for the in-situ remediation of contaminated soil in natural ecosystems, which has broad application prospects.

[0028] Unless otherwise specified, the experimental methods, materials and reagents used in the following examples are all conventional methods.

[0029] Example 1:

[0030] (1) Enrichment culture of the strain

[0031] The anaerobic sludge sample was collected near the sewage outlet of the oil production sewage treatment plant in Liaohe Oilfield (Panjin City) in November 2022. The tools used for sampling were a 1-meter soil drill and a 1L reagent bottle sterilized at high temperature. The soil drill was inserted deep into the sludge, and the taken sludge was quickly sent into the 1L reagent bottle. River water was added to remove the air in the bottle. After removing the air, the bottle was sealed with a suspension cover. The collected sample was placed in a heat preservation box and immediately taken back to the laboratory for storage in a -20°C refrigerator.

[0032] Under the condition of nitrogen filling, 10.00 g of the sludge soil sample was weighed into a 100 mL serum bottle, 1 mL of ferric citrate suspension (with an iron content of about 1 g / L) and 50 mL of deionized water were added, and it was left to stand in the dark at 30°C for 1 week for recovery culture. The cultured soil suspension was centrifuged at 5000 r·min -1 for 10 min under an anaerobic glove box, and the supernatant was taken as the microbial enrichment culture.

[0033] (2) Isolation of the strain Z1 with iron reduction function

[0034] Six anaerobic sterile serum bottles containing 20 mL of ferric citrate liquid medium were taken, placed on a fixed rack and numbered 1-6, and then placed in a sterile operating table. 1 mL of the enrichment culture solution was injected into the No. 1 serum bottle with a disposable sterile syringe, and 9 mL of sterile water cooled to room temperature was added and mixed evenly. The dilution degree was 10 -1 . 9 mL of sterile water cooled to room temperature was successively added to the No. 2-6 serum bottles. 1 mL of the bacterial solution in the No. 1 serum bottle was taken into the No. 2 serum bottle. At this time, the dilution degree was 10 -2 . According to this operation, it was successively diluted to 10 -6Absorb an appropriate amount of bacterial liquid with different dilution degrees into the ferric citrate solid medium, and spread it evenly with a disposable sterile spreading rod. Set two groups for each dilution gradient. Place the ferric citrate solid medium in an anaerobic jar and culture it under dark conditions at 30 °C for 3 - 4 days. Then, use a disposable sterile syringe to pick a single colony from the ferric citrate solid medium into the ferric citrate liquid medium for continued culture. If the ferric citrate liquid medium changes from yellow to green, it is an iron-reducing bacterium. To further purify the strain, dilute the liquid of the discolored medium and add it to the ferric citrate solid medium, and repeat the above steps 6 times to obtain a single strain Z1.

[0035] Ferric citrate liquid medium formula: The main components of the medium are FeC6H5O7 2.45 g / L, C3H5O3Na 1.12 g / L, NaCl 1.0 g / L, MgCl2·6H2O 0.5 g / L, KH2PO4 0.2 g / L, NH4Cl 0.3 g / L, KCl 0.3 g / L, CaCl2·2H2O 0.015 g / L, FeCl2·4H2O 1.5 mg / L, CoCl2·6H2O 190 μg / L, MnCl2·4H2O 100 μg / L, ZnCl2 70 μg / L, H3BO3 6 μg / L, Na2MoO4·2H2O 36 μg / L, NiCl2·6H2O 24 μg / L, CuCl2·2H2O 2 μg / L, Na2SeO3·5H2O 6 μg / L, Na2WO4·2H2O 8 μg / L. Add sodium resazurin 0.025% (w / v). After boiling the medium to remove oxygen, add L-cysteine 24 mg / L and Na2S·9H2O 48 mg / L as reducing agents to further remove oxygen in the medium. Add 30 mM of NaHCO3 buffer reagent, and adjust the pH of the medium to 7.3 with a CO2 and N2 mixed gas. After sterilization and cooling, add a vitamin mixture filtered and sterilized through a 0.22 μm filter membrane, and its composition is as follows: vitamin B2 50 μg / L, lipoic acid 50 μg / L, vitamin B 20 μg / L, vitamin B6 100 μg / L, vitamin H 20 μg / L, vitamin B5 50 μg / L, nicotinic acid 50 μg / L, vitamin B1 50 μg / L, p-aminobenzoic acid 50 μg / L.

[0036] Ferric citrate solid medium: It is prepared by adding 20 g / L of agar to the ferric citrate liquid medium.

[0037] (3) Classification and identification of strain Z1

[0038] 16S rRNA gene sequence homology analysis: The bacterial solution of strain Z1 after isolation and purification was sequenced. The specific steps were as follows: The bacterial solution of the isolated and purified strain Z1 was filtered on a filter membrane, the total DNA on the filter membrane was extracted, and this was used as the gene amplification template for polymerase chain reaction (PCR). The universal primers were 27F and 1492R for PCR reaction, and Sanger sequencing was performed therefrom. The sequencing results were compared with the National Center for Biotechnology Information (NCBI) database to obtain the strain similarity results.

[0039] The results showed that the homology comparison of the 16S rRNA of strain Z1 was higher than 99% with the microorganisms under the genus Citrobacter, and the homology with the strain Citrobacter amalonaticus was the highest. The 16S rRNA gene sequence of strain Z1 was as follows:

[0040] CAGCTACACATGCAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTT

[0041] GCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCCGAT

[0042] GGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAATGTCGC

[0043] AAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCC

[0044] AGATGGGATTAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGACGA

[0045] TCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACA

[0046] CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATG

[0047] GGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCG

[0048] GGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGGTTAAGGTTAATAAC

[0049] CTTAGCCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTG

[0050] CCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTAC

[0051] TGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATC

[0052] CCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTTGAGTC

[0053] TCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAG

[0054] ATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTG

[0055] ACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG

[0056] GTAGTCCACGCCGTAAACGATGTCTATTTGGAGGTTGTGCCCTTGAGG

[0057] CGTGGCTTCCGGAGCTAACGCGTTAAATAGACCGCCTGGGGAGTACG

[0058] GCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG

[0059] GTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGT

[0060] CTTGACATCCACAGAACTTGGCAGAGATGCCTTGGTGCCTTCGGGAA

[0061] CTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATG

[0062] TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGC

[0063] GGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGG

[0064] AAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTA

[0065] CACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAG

[0066] CAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAAC

[0067] TCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCA

[0068] CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGG

[0069] GAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCT

[0070] ACCACTT

[0071] Phylogenetic tree constructed by homology alignment of 16S rRNA gene sequences of strain Z1 and related strains is shown in Figure 1 .

[0072] The strain is Citrobacter amalonaticus Z1, which was deposited at the China Center for Type Culture Collection on October 12, 2023, with the deposit number CCTCC NO: M 20231863.

[0073] Example 2: Optimal growth conditions of strain Z1

[0074] (1) Determination of the optimal pH for the growth of strain Z1

[0075] Adjust the pH value of the LB liquid medium to 4, 7.5, and 9 with 0.1 mol / L NaOH and HCl. After sterilization, inoculate 1 mL of the obtained bacterial suspension into 100 mL of anaerobic sterilized LB liquid medium, and place it in a 30 °C incubator for dark cultivation. Then, take out 200 μL from the conical flask with a sterile syringe at 0, 5, 10, 25, 35, 48, 72 h, and 96 h respectively, place it in a 96-well plate, and use a microplate reader (Recan Spark 10M) to measure the OD of the bacterial solution. 600 value. The formula of the LB medium is tryptone 10 g / L, yeast extract powder 5 g / L, and NaCl 10 g / L.

[0076] The results show (see Figure 2 ) that strain Z1 grows faster at pH = 7.5 and pH = 9, and grows fastest at pH = 7.5. At pH = 4, it starts to grow rapidly after 40 hours. It can be seen that the most suitable pH value for strain Z1 is 7.5.

[0077] (2) Determination of the optimal growth temperature of strain Z1

[0078] Adjust the pH value of the LB liquid medium to 7 with 0.1 mol / L NaOH. After sterilization, inoculate 1 mL of the above bacterial suspension into 100 mL of anaerobic sterilized LB liquid medium, and place it in 4 °C, 25 °C, and 50 °C incubators for dark cultivation respectively. Then, take out 200 μL from the conical flask with a sterile syringe at 0, 5, 10, 25, 35, 48, 72, and 96 h respectively, place it in a 96-well plate, and use a microplate reader to measure the OD of the bacterial solution. 600 value. The formula of the LB medium is the same as that in the above experiment.

[0079] The results show (see Figure 3 ) that strain Z1 grows fastest at 25 °C and hardly grows at 50 °C. Among 4 °C and 25 °C, the growth rate is the fastest from 0 to 20 hours.

[0080] Example 3: Screening of the most suitable carbon source during the iron reduction process of strain Z1

[0081] The experimental system was a 100 mL anaerobic bottle. 80 mL of iron citrate liquid medium without added carbon source was added to the 100 mL anaerobic bottle. After injecting a mixture of 80% N2 and 20% CO2 into the bottle for half an hour, it was stoppered with an aluminum cap and sterilized at 121 °C for 30 minutes. After sterilization and cooling, 1 mL of pre-sterilized 1 mol / L glucose, sucrose, sodium lactate, and sodium acetate was injected into the medium using a disposable sterile syringe, and then 1 mL of the bacterial suspension of strain Z1 that had been cultured for 24 hours in advance was injected using a disposable sterile syringe. It was then placed in a 30 °C constant temperature incubator for cultivation. On the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th, and 10th days, 5 mL of the medium liquid was extracted using a disposable sterile syringe for analyzing the ferrous iron concentration.

[0082] The bacterial suspension was the bacterial suspension of the single colony Citrobacter amalonaticus Z1 obtained in Example 1 above that had been cultured for 24 hours in advance (i.e., prepared from an iron citrate solid medium).

[0083] Determination of iron reduction ability: Take 1 mL of the culture solution in a 10 mL centrifuge tube, add an acetic acid - ammonium acetate buffer solution and a 0.5% phenanthroline color reagent. After color development for 15 minutes, measure the absorbance value at 510 nm using a UV - visible spectrophotometer (Shimadzu UV - 2550).

[0084] As shown in the results (see Figure 4 ), the ferrous iron of all four carbon sources was increasing. Among them, the ferrous iron of sucrose and glucose increased more. However, because the sugars decomposed from sucrose and glucose are reducing, more ferrous iron participated in the chemical reaction for reduction. Therefore, sodium lactate, which is relatively stable, was selected as the carbon source for subsequent experiments in cultivation.

[0085] Example 4: Degradation of petroleum hydrocarbons (n - hexadecane) by strain Z1 and its iron reduction ability

[0086] The experimental system was a 100 mL anaerobic bottle. 80 mL of iron citrate liquid medium was added to the 100 mL anaerobic bottle. After injecting a mixture of 80% N2 and 20% CO2 into the bottle for half an hour, it was stoppered with an aluminum cap and sterilized at 121 °C for 30 minutes. After sterilization and cooling, 1 mL of the bacterial suspension of strain Z1 that had been cultured for 24 hours in advance as described in Example 3 was injected into the medium using a disposable sterile syringe. Finally, a certain amount of pre - prepared pollutant mother liquor was added to make the initial concentration of n - hexadecane in the anaerobic bottle 500 ppm. It was then placed in a 30 °C constant temperature incubator for cultivation. On the 0th, 5th, 10th, 15th, 20th, 30th, and 40th days of the experiment, 12 mL of the medium liquid was extracted using a disposable sterile syringe for analyzing the ferrous iron concentration and the pollutant concentration. Two parallels were set up for the experiment.

[0087] The pollutant mother liquor was prepared by dissolving n-hexadecane in n-hexane, and the concentration of n-hexadecane in the mother liquor was 40 g / L. 1 mL of the pollutant mother liquor was added to the ferric citrate liquid medium.

[0088] Determination of iron reduction ability: The same as in Example 3.

[0089] Extraction of n-hexadecane: Transfer all 5 mL of the culture solution to a 30 mL separatory funnel, measure 5 mL of dichloromethane and add it to the separatory funnel, shake and extract for 5 min, let it stand for 10 min, wait for the two phases to separate, collect the lower organic phase, then add 5 mL of dichloromethane, repeat the above operation 2 times, and combine the extraction solutions.

[0090] Concentration operation of n-hexadecane: Use a parallel evaporator (BUCHI Syneore) for the extraction solution. The instrument conditions are a water bath temperature of 40 °C and a vacuum degree of 529 mbar, concentrate to 1 mL, then add 5 mL of n-hexane, with the conditions of a water bath temperature of 40 °C and a vacuum degree of 184 mbar, concentrate to 1 mL, add 5 mL of n-hexane again, with the conditions of a water bath temperature of 40 °C and a vacuum degree of 184 mbar, and concentrate to dryness. Then use chromatographic-grade n-hexane to make the volume of the extracted n-hexadecane solution constant, filter through an organic filter membrane, and store it in a gas chromatography sample bottle for subsequent detection.

[0091] Detection conditions for n-hexadecane: The GC-MS instrument (Thermo Scientific ISQ) uses a non-polar capillary column (TR-1MS, 30 m × 0.25 mm × 0.25 μm) for operation. The sample detection conditions are: an initial temperature of 40 °C, lasting for 3 min; rising to 250 °C at a rate of 10 °C·min -1 and lasting for 1 min; rising to 290 °C at a rate of 5 °C·min -1 and lasting for 20 min. The ion source temperature of the instrument is 290 °C, the transfer line temperature is 290 °C, and the injection port temperature is 280 °C. The splitless injection method is adopted, and the injection volume is 1.0 μL; the EI ion source energy is maintained at 70 ev, and the ion fragment mass scanning range is 30 - 300 amu. The carrier gas is 99.99% high-purity helium gas, and the carrier gas flow rate is 1.0 mL·min -1 .

[0092] As shown in the results (see Figure 5 ), during the 40-day culture process of the strain Citrobacter amalonaticus Z1, using sodium lactate and n-hexadecane as substrates, the n-hexadecane was degraded from 479.46 mg / L to 77.45 mg / L, and the degradation rate was 10.05 mg / (L·d).

[0093] The results show (see Figure 6) During the 40-day cultivation process of the strain Citrobacter amalonaticus Z1, using sodium lactate and ferric citrate as substrates, ferric iron was reduced to ferrous iron. The concentration of ferrous iron increased from 32.4 mg / L to 323.5 mg / L, with a growth rate of 7.28 mg / (L·d). Example 5: Determination of the degradation performance of soil petroleum hydrocarbons and iron reduction ability in soil by strain Z1 under anaerobic conditions

[0094] The experimental system was a 300 mL anaerobic bottle. In an anaerobic glove box, soil was prepared by adding aged n-hexadecane. The concentration of n-hexadecane in the soil was 1% (v / w). Then, 150 mL of sterile and deoxygenated ultrapure water was added. 8 mL of the bacterial suspension of the strain Citrobacter amalonaticus Z1 cultivated 24 hours in advance as described in Example 3 above was injected using a disposable sterile syringe. After covering the bottle with a stopper and lid, it was taken out of the glove box and cultured in an incubator at 30 °C. 10 g of soil samples were collected at 0, 5, 10, 15, 20, 30, and 40 days of the experiment for determining the concentrations of different forms of ferrous iron and n-hexadecane in the soil.

[0095] The soil prepared with the aged n-hexadecane had an n-hexadecane concentration of 1% (v:w), that is, 1 mL of n-hexadecane was added to 100 g of soil.

[0096] Extraction and determination of ferrous iron forms in soil: Weigh 1.00 g of soil that has been freeze-dried and passed through a 60-mesh sieve into a 50 mL centrifuge tube. Add 8 mL of 1 mol / L MgCl2 (pH 7), shake at 220 rpm for 2 hours, centrifuge at 8000 rpm / min for 5 min in a centrifuge, take the supernatant, filter it through a 0.45 μm water-based filter membrane, and then add it to a centrifuge tube pre-added with 10 μL of 6M HCl for testing the concentration of ion-exchangeable Fe(II); add 8 mL of 1 mol / L HCl to the centrifuged precipitate, shake at 220 rpm for 48 hours, centrifuge at 8000 rpm / min for 5 min, take the supernatant, filter it through a 0.45 μm water-based filter membrane, and use it to test Fe(II) in the amorphous form (surface adsorbed state, carbonate-bound state, poorly crystalline iron oxides, sulfides, hydroxides); add 8 mL of 6 mol / L HCl to the centrifuged precipitate, shake at 220 rpm for 24 hours, centrifuge at 8000 rpm / min for 5 min, take the supernatant, filter it through a 0.45 μm water-based filter membrane, and use it to test the active Fe(II) in the mineral structural state of the soil except for pyrite; finally, add 8 mL of 16 mol / L HNO3 to the centrifuged precipitate, shake at 220 rpm for 2 hours, centrifuge at 8000 rpm / min for 5 min, take the supernatant, filter it through a 0.45 μm water-based filter membrane, and use it to test Fe(II) in pyrite.

[0097] Determination of n - hexadecane in soil: Add 2 g of diatomaceous earth into a 40 mL extraction cell, then add 1.00 g of freeze - dried soil passed through a 60 - mesh sieve and 2 g of quartz sand. Finally, add 2 g of diatomaceous earth on the top and extract with a Buchi rapid extractor. The extraction temperature is 100 °C, the extraction pressure is 100 bar, and n - hexane and acetone are mixed in a ratio of 1:1 (V / V) as the extraction solvent. The operating program of the extractor is as follows: heating time 5 min, holding time 5 min, discharging time 2 min, circulating 2 times, cleaning 1 min, and nitrogen purging 2 min. After extraction, completely evaporate the n - hexane and acetone 1:1 (V / V) mixed solvent through a Buchi parallel evaporator, then elute C16 with 5 mL of chromatographically pure n - hexane, and conduct subsequent detection on a GC - MS instrument (Thermo Scientific ISQ).

[0098] As shown in the results (see Figure 7 ), during the 40 - day cultivation process, the total Fe(II) in the soil added with strain Citrobacter amalonaticus Z1 increased from 3718.4 mg / L to 5581.6 mg / L, and the growth rate was 46.58 mg / (L·d).

[0099] As shown in the results (see Figure 8 ), during the 40 - day cultivation process, the n - hexadecane in the soil added with strain Citrobacter amalonaticus Z1 degraded from 350.16 mg / L to 234.25 mg / L, and the degradation rate was 2.89 mg / (L·d).

[0100] As shown in the results (see Figure 9 ), during the 40 - day cultivation process, the ion - exchangeable Fe(II) in the soil added with strain Citrobacter amalonaticus Z1 was not detected. The ratio of amorphous Fe(II) to the total soil Fe(II) increased, the relative ratio of reactive Fe(II) decreased, and the Fe(II) in pyrite accounted for less of the total soil Fe(II).

[0101] Example 6: Preparation of Bacterial Agent

[0102] Prepare a bacterial agent for degrading petroleum hydrocarbon pollutants from the preserved strain. Using the ferric citrate medium as the base, inoculate 3% Citrobacter amalonaticus Z1 and incubate in the dark and statically at pH = 7.5 and a temperature of 25 °C for 5 days. Then, centrifuge at 8000 r·min -1Centrifuge for 10 min to collect the cells of strain Z1, and add glycerol or other solid and liquid carriers to the cells to obtain a culture concentrate of Citrobacter amalonaticus Z1, which is the microbial agent for degrading petroleum hydrocarbon pollutants as shown. This microbial agent can be added to petroleum hydrocarbon-polluted water bodies or soil environments, and has broad application prospects for bioremediation.

Claims

1. An iron-reducing strain capable of degrading petroleum hydrocarbons under anaerobic conditions, characterized in that: The strain is Citrobacter amalonaticus Z1, which uses petroleum hydrocarbons as an electron donor. It was deposited at the China Center for Type Culture Collection on October 12, 2023, with the deposit number CCTCC NO: M20231863.

2. Use of the iron-reducing strain for degrading petroleum hydrocarbons according to claim 1, characterized in that: The application of the strain in iron reduction reaction.

3. Use of the iron-reducing strain for degrading petroleum hydrocarbons according to claim 1, characterized in that: The application of the strain in degrading petroleum hydrocarbons in the environment.

4. Use of the iron-reducing strain for degrading petroleum hydrocarbons according to claim 3, characterized in that: The application of the strain in degrading petroleum hydrocarbons in soil or groundwater under anaerobic conditions, using ferric iron as an electron acceptor and small-molecule straight-chain or branched-chain alkanes as electron donors.

5. A microbial agent, characterized in that: The bacterial agent contains Citrobacter amalonaticus Z1 described in claim 1.

6. The bacterial agent according to claim 5, wherein: The bacterial agent contains the cells of Citrobacter amalonaticus Z1, the culture, the concentrate of the culture, or the bacterial suspension of the culture.

7. Use of the microbial agent according to claim 5, characterized in that: The application of the bacterial agent in degrading petroleum hydrocarbon pollutants in environmental media with ferric iron as an electron acceptor under anaerobic conditions.

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